We are continuing our study of the E. Coli bacteriophage T4 model system for duplex DNA replication in which efficient DNA replication in vitro is achieved with purified proteins encoded by T4 phage: T4 DNA polymerase (gene 43), gene 32 DNA helix-destabilizing protein, the gene 44/62 and gene 45 polymerase accessory proteins, the genes 41, 61, and 59 primase-helicase, RNase H, and DNA ligase. We are collaborating with Tim Meuser and Craig Hyde, NIAMS, to determine the structure of the T4 DNA replication proteins by X-ray diffraction. Our recent work has been directed at determining how primer removal by T4 RNase H is coordinated with other steps in lagging and leading strand synthesis, understanding what controls how much DNA is removed along with the RNA primer, and elucidating the structure and hydrolytic mechanism of the nuclease. T4 RNaseH is a 5' to 3' exonuclease that removes RNA primers from the lagging strand of the DNA replication fork, and is a member of the RAD2 family of eukaryotic and prokaryotic replication and repair nucleases. In the crystal structure, the most conserved acidic residues are clustered together surrounding two Mg in a cleft wide enough for single DNA. Site-directed mutagenesis indicates that only one of the two Mg is essential for catalysis, and that the disordered region above the cleft is involved in DNA binding. T4 gene 32 protein converts T4 RNase H into a processive exonuclease, but inhibits its flap endonuclease, suggesting an important role for 32 protein in controlling T4 RNase H degradation of RNA primers and adjacent DNA during each lagging strand cycle. We are studying the mechanism by which the gene 59 protein stimulates DNA unwinding by the 41 helicase, and primer synthesis dependent on both the 41 and 61 proteins. Solution of the crystal structure of the 59 helicase assembly protein is in progress.